recommended methods for the identification and analysis of piperazines in seized materials (2013)
TRANSCRIPT
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Recommended methods for the
Identifcation and Analysis oPiperazines in Seized Materials
Manual for use by national drug analysis laboratories
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Photo credits:UNODC Photo Library; UNODC/Ioulia Kondratovitch; Alessandro Scotti.
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Lby Sf S
United nationS oice on drUgS and crime
V
Recommended Methods or
the Identifcation and Analysis o
Piperazines in Seized Materials
MANUAL FOR USE BY
NATIONAL DRUG ANALYSIS LABORATORIES
UNITED NATIONS
New York, 2013
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ST/NAR/47
Original language: English United Nations, January 2013. All rights reserved, worldwide.
The designations employed and the presentation o material in this publication donot imply the expression o any opinion whatsoever on the part o the Secretariato the United Nations concerning the legal status o any country, territory, city orarea, or o its authorities, or concerning the delimitation o its rontiers or boundaries.Mention o names o rms and commercial products does not imply the endorse-
ment o the United Nations.
This publication has not been ormally edited.
Publishing production: English, Publishing and Library Section, United NationsOce at Vienna.
Note
Operating and experimental conditions are reproduced rom the original reerence
materials, including unpublished methods, validated and used in selected nationallaboratories as per the list o reerences. A number o alternative conditions andsubstitution o named commercial products may provide comparable results in manycases, but any modication has to be validated beore it is integrated into laboratoryroutines.
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iii
Acknowledgements
UNODCs Laboratory and Scientic Section (LSS), headed by Dr. Justice Tettey,
wishes to express its appreciation and thanks to Ms. Pamela Smith or the prepara-tion o the rst drat o this manual.
LSS would also like to thank Mr. Jeery Comparin o the United States DrugEnorcement Administration, Mr. Paul Loo, Mr. Chad Mehaux and Ms. ValrieBlisle o the Canadian Border Services Agency and Dr. Mark Baron o theUniversity o Lincoln, United Kingdom or their expert reviews and valuablecontribution.
The preparation o this manual was coordinated by Dr. Conor Crean and the con-tributions o UNODC sta and interns, Ms. Bridgette Webb and Mr. Diego Pazosare grateully acknowledged.
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Contents
Page
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Purpose and use o the Manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. General aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.1 Description o the pure compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.2 Licit uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2.3 Control status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82.4 Illicit products/use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82.5 Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3. Illicit manuacture o piperazines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.1 Illicit manuacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
4. Qualitative and quantitative analysis o materials containing piperazines . . 12
4.1 Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124.2 Solubility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124.3 Screening tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134.4 Microcrystalline tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174.5 Tin-layer chromatography (LC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194.6 Gas chromatography (GC) with fame ionization detection (GC-FID) 234.7 Gas chromatography-mass spectrometry (GC-MS) . . . . . . . . . . . . . . . . 254.8 Gas chromatography-inrared detection (GC-IRD) . . . . . . . . . . . . . . . . 284.9 High pressure liquid chromatography (HPLC) . . . . . . . . . . . . . . . . . . . . 294.10 Capillary electrophoresis (CE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324.11 Fourier transorm inrared (FIR) spectroscopy . . . . . . . . . . . . . . . . . . 33
5. Library inormation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
5.1 Ultraviolet (UV) spectrophotometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355.2 GC-MS data or selected piperazines . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Additional reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37Reerences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
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1. Introduction
1.1 Background
Piperazine, a heterocyclic six-membered ring compound which contains two nitro-gens in the 1 and 4 positions, is a cyclic member o the ethylenediamine group omolecules [1, 2]. The abuse o substituted derivatives o piperazine was rst reportedin the United States in 1996 and has, since then, spread to a number o countriesworldwide [3]. The large scale use o synthetic derivatives o piperazine as substi-tutes or mimics o ecstasy started in New Zealand in the early 2000s and becamecommon in Europe ater 2004 [4].
The rst piperazine derivative encountered was 1-benzylpiperazine (BZP), one o a
group o phenyl and benzyl substituted piperazines that have become prevalentworldwide, especially in traditional 3,4-methylenedioxymethamphetamine (MDMA)markets. Other widely used piperazines include 1-(3-chlorophenyl)piperazine,(mCPP) and 1-(3-trifuoromethylphenyl)piperazine (TFMPP), the latter o which iscommonly ound in combination with BZP.
BZP itsel is a central nervous system stimulant with a potency o 10% that od-ametamine [4]. It has been reported to stimulate the release o dopamine,noradrenaline, and serotonin, and also inhibit their reuptake. The substances are thusametamine mimics and predominantly ound in tablet orm either alone, in com-bination with other piperazines or with ametamine, cocaine, ketamine or MDMA.
Neither BZP nor any other substituted piperazine are listed in the Schedules o theUnited Nations 1971 Convention on Psychotropic Substances. However, in 2007,the International Narcotics Control Board (INCB) requested the World HealthOrganization (WHO) to consider reviewing piperazine derived compounds or pos-sible scheduling under the 1971 Convention. Independently, many countries haveintroduced legislation controlling the use o BZP. This includes the United Statesand the countries o the European Union (EU), which submitted BZP or EU control
in 2008 ollowing a risk assessment using the early warning system o the EuropeanMonitoring Centre or Drugs and Drug Addiction (EMCDDA) [5].
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2 Recommended methods or the identifcation and analysis o piperazines in seized materials
1.2 Purpose and use o the Manual
The presentManual is one in a series o similar publications dealing with the iden-
tication and analysis o various classes o drugs under international control. Thesemanuals are the outcome o a programme pursued by UNODC since the early 1980s,aimed at the harmonization and establishment o recommended methods o analysisor national drug analysis laboratories.
This Manual was prepared taking into account the Commission on Narcotic Drugs2012 resolution: 55/1 Promoting international cooperation in responding to thechallenges posed by new psychoactive substances, which encourages the UnitedNations Oce on Drugs and Crime and other relevant international organizations,
upon request, to provide Member States with technical assistance, including bysupporting orensic and toxicological capability, to respond to the challenges posedby new psychoactive substances.
In accordance with the overall objective o the series, the present manual suggestsapproaches that may assist drug analysts in the selection o methods appropriate orthe sample under examination and provide data suitable or the purpose at hand,leaving room also or adaptation to the level o sophistication o dierent laboratoriesand various legal requirements. The majority o methods included in this manualare validated, methods which have been used or a number o years in reputable
laboratories and as part o inter-laboratory studies, collaborative exercises and pro-ciency tests. The reader should be aware, however, that there are a number o othermethods, including those published in the orensic science literature, which mayalso produce acceptable results. Any new method that is to be used in the readerslaboratory must be validated and/or verifed prior to routine use.
In addition, there are a number o more sophisticated approaches, but they may notbe necessary or routine operational applications. Thereore, the methods describedhere should be understood as guidance, that is, minor modications to suit local
circumstances should not aect the validity o the results. The choice o the meth-odology and approach to analysis, as well as the decision whether or not additionalmethods are required, remain with the analyst and may also be dependent on theavailability o appropriate instrumentation and the level o legally acceptable prooin the jurisdiction within which the analyst works.
Attention is also drawn to the vital importance o the availability to drug analystso reerence materials and books on drugs o abuse and the analytical techniquesused or their identication. Moreover, the analyst must o necessity keep abreasto current trends in drug analysis, consistently ollowing current analytical and oren-
sic science literature.
UNODCs Laboratory and Scientic Section would welcome observations on thecontents and useulness o the present Manual. Comments and suggestions may beaddressed to:
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Recommended methods or the identifcation and analysis o piperazines in seized materials 3
Laboratory and Scientic SectionUnited Nations Oce on Drugs and CrimeVienna International Centre
P.O. Box 5001400 ViennaAustriaFax: (+43-1) 26060-5967E-mail: [email protected]
All manuals, as well as guidelines and other scientic-technical publications, maybe requested by contacting the address above.
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2. General aspects
2.1 Description o the pure compounds.
The ollowing table presents the structures and selected data or the three most commonlyencountered piperazines. A comprehensive list o piperazines is provided in table 2.
Table 1. Description o most common piperazines
1-Benzylpiperazine (BZP) Empirical formula: C11
H16
N2
CAS No.: 2759-28-6
Molecular weight: 176.26 g/mol
Refractive index: 1.5470Density: 1.014 g/ml
Physical appearance: clear to yellowish liquid
1-(3-Triuoromethylphenyl)
piperazine (TFMPP)
Empirical formula: C11
H13
F3N
2
CAS No.: 15532-75-9
Molecular weight: 230.23 g/mol
Refractive index: 1.521
Density: 1.226 g/ml
Physical appearance: white powder
1-(3-Chlorophenyl)piperazine
(mCPP)
Empirical formula: C10
H13
ClN2
CAS No.: 6640-24-0
Molecular weight: 196.68 g/molRefractive index: 1.598-1.600
Density: 1.19 - 1.195 g/ml
Physical appearance: clear to yellowish liquid
N
NH
N NH
F3C
N NH
Cl
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Table2.
Che
micalstructuresanddescriptionofselectedpiperaz
ines
Commonname
Abbreviation
CASnumber
R1
R2
Piperazine
110-85-0
H
H
1-Benzylpiperazine
BZP
2759-28-6
Ph-CH2
H
1-Benzyl-4-me
thylpiperazine
MBZP
374898-00-7
Ph-CH2
CH3
1,4-Dibenzylpiperazine
DBZP
1034-11-3
Ph-CH2
C7
H7
1-(3-Thienylmethyl)piperazine
3-TMP
130288-91-4
C5
H5
S
H
1-(2-Phenyleth
yl)piperazine
2-PEP
5321-49-3
Ph-CH2-CH2
H
1-(3,4-Methyle
nedioxybenzyl)piperazine
MDBZP
55827-51-5
3
,4-methylenedioxybenzyl
H
C
ommonname
Abbreviation
CASnumber
R1
R2
R3
R4
R5
1-(2-Methoxyp
henyl)piperazine
2-MeOPP/oMeOPP
35386-24-4
OCH3
H
H
H
H
1-(3-Methoxyp
henyl)piperazine
3-MeOPP/mMeOPP
16015-71-7
H
OCH3
H
H
H
N
N
R2
R
N
N
R1
R2
R3
R4
R5
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Recommended methods or the identifcation and analysis o piperazines in seized materials 7
1-(4-Methoxyp
henyl)piperazine
4-MeOPP/pMeOPP
38212-30-5
H
H
OCH3
H
H
1-(2-Trifuorom
ethylphenyl)piperazine
oTFMPP
3854-31-9
CF3
H
H
H
H
1-(3-Trifuorom
ethylphenyl)piperazine
TFMPP/mTFMPP
15532-75-9
H
CF3
H
H
H
1-(4-Trifuorom
ethylphenyl)piperazine
pTFMPP
30459-17-7
H
H
CF3
H
H
2-Methylphen
ylpiperazine
2-MePP/oMePP
39512-51-1
CH3
H
H
H
H
3-Methylphen
ylpiperazine
3-MePP/mMePP
41186-03-2
H
CH3
H
H
H
4-Methylphen
ylpiperazine
4-MePP/pMePP
39593-08-3
H
H
CH3
H
H
1-(4-Bromo-2,5-dimethoxybenzyl)
piperazine
2C-BBZP
1094424-37-9
OCH3
H
Br
OCH3
H
1-(2-Chlorophenyl)piperazine
2CPP/oCPP
41202-32-8
Cl
H
H
H
H
1-(3-Chlorophenyl)piperazine
mCPP
6640-24-0
H
Cl
H
H
H
1-(4-Chlorophenyl)piperazine
4-CPP/pCPP
38212-33-8
H
H
Cl
H
H
1-(2-Fluorophenyl)piperazine
2-FPP/oFPP
1011-15-0
F
H
H
H
H
1-(4-Fluorophenyl)piperazine
4-FPP/pFPP
2252-63-3
H
H
F
H
H
1-(2,3-Dimethylphenyl)piperazine
2,3-XP
1013-22-5
CH3
CH3
H
H
H
1-(3,4-Dimethylphenyl)piperazine
3,4-XP
1014-05-7
H
CH3
CH3
H
H
1-(2,5-Dimethylphenyl)piperazine
2,5-XP
1013-25-8
CH3
H
H
CH3
H
1-(2,4-Dimethylphenyl)piperazine
2,4-XP
1013-76-9
CH3
H
CH3
H
H
1-(3-Chlorophenyl)-4-(3-chloropropyl)
piperazine
mCPCPP
39577-43-0
H
Cl
H
H
C3
H6
Cl
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8 Recommended methods or the identifcation and analysis o piperazines in seized materials
2.2 Licit uses
1-Benzylpiperazine (BZP) and the other substituted piperazines listed in tables 1
and 2 have no current human or veterinary pharmaceutical use in any country,although piperazine itsel is used as an anthelmintic drug. Piperazine derivatives
serve as precursors or intermediates in the synthesis o many pharmaceutically active
compounds, including ciprofaxin, the quinolone antibiotics, phenothiazines, silde-
nal, tadalal and antihelminthics [6, 7, 8, 9].
O the substituted piperazines that have been used illicitly, mCPP is an synthetic
precursor in the production, and an active metabolite, o the anti-depressants trazo-
done, neazodone and etoperidon [10, 11]. 1-(3,4-methylenedioxybenzyl)piperazine
(MDBZP) is a metabolite o the withdrawn nootropic drug pexide and 1-(4-meth-oxyphenyl)piperazine (MeOPP) is a known metabolite o a number o prescribed
drugs including enciprazione, milipertine and urapidil [11].
2.3 Control status
None o the benzyl or phenyl substituted piperazines covered in thisManual are listed
in the Schedules o the United Nations 1971 Convention on Psychotropic Substances.
However, many countries have introduced national control measures or some pipera-zines. For example, BZP was classied as a schedule 1 controlled substance in the
United States in 2002, while the European Monitoring Centre or Drugs and Drug
Addiction (EMCDDA) submitted BZP or EU control ollowing completion o a risk
assessment in 2008. 1-(3-Chlorophenyl)piperazine (mCPP) is not controlled interna-
tionally because it is used in drug synthesis, and has also not been submitted or risk
assessment under the EU system, although a number o European countries have
independently implemented measures or its control [12]. During a recent meeting
o the WHO Expert Committee on Drug Dependence, several members o the pipe-
razine amily were pre-reviewed (BZP, TFMPP, mCPP, MeOPP and MDBZP) [11].
2.4 Illicit products/use
The bulk powders used in ormulations o piperazines are readily available rom
commercial suppliers in both China and India. The bulk material is then cut with
sugars and/or other drugs prior to processing into capsules and tablets, which are
similarly priced to ecstasy. BZP is most oten encountered as o-white or coloured
tablets, which oten bear imprints similar to those seen on MDMA tablets, and
indeed the tablets are oten sold as ecstasy. Typical concentrations o BZP in theseormulations range rom 50-200 mg. The concentration mCPP in seizures o tablets
has been reported to be in the range 90-110 mg [13]. Seizures are oten ound to
contain a mixture o substituted piperazines cut with caeine, and oten contain
controlled substances such as MDMA, ketamine or ametamine [4,14].
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The piperazines are usually ingested as tablets or capsules. However with prolongeduse, a more rapid drug response is oten desired and this is usually achieved bysmoking, snorting, or more rarely by injection. Snorting and injection have unpleas-
ant side eects such as burning o the nasal passages with the ormer and a burningsensation with the latter route. These eects are a result o the typically very causticnature o the piperazine ormulations (pH o 12). For this reason, alcohol or someother drug is commonly used in conjunction with the piperazine to minimize theseadverse eects. This class o drugs seems to attract a signicant population o newdrug users and this may be due to the perception that it is a sae/legal drug choice.Users o substituted piperazines seem to be more apt to use multi-drug cocktailsthan MDMA users, with alcohol, cannabis and synthetic cannabinoids being themost commonly reported drugs used in combination [8, 15, 16, 17].
2.5 Pharmacology
The majority o pharmacological studies o piperazines have ocused on BZP andhave indicated that it mimics the behaviour o d-ametamine, with 10% o itspotency. BZP has been reported to exhibit a potential or abuse and dependencesimilar to that o amphetamine, and causes a stimulus-like eect, increasing heartrate and systolic blood pressure. Furthermore, results rom animal studies demon-strate that this compound stimulates the release, and inhibits the reuptake, o dopa-mine, serotonin (5-HT) and noradrenaline [17].
Research on mixtures o BZP and TFMPP (conducted because they are requentlyound in combination) showed the release o both dopamine and serotonin viamechanisms dependent on their transporters [18]. Combinations o BZP and TFMPP,in proportions ranging rom 2:1 to 10:1, have been reported to mimic the molecularmechanism o MDMA, causing similar entactogenic body eects and thereore mak-ing it a popular MDMA substitute [17, 19, 20].
While there are a lack of detailed studies concerning many of the piperazine deriva-tives, there has been some research on mCPP, MDBZP and pMeOPP, though mostly
on metabolism rather than toxicological effects [21]. It has been reported that sero-
tonin syndrome, which induces symptoms such as anxiety, dizziness, confusion,
shivering and sensitivity to light and noise, can develop following mCPP consump-
tion [4]. In animal studies,high doses of BZP/TFMPP have been observed to cause
seizures in rats. In humans, a high rate of adverse reactions, including severe toxicity
and seizures, to the consumption of BZP/TFMPP pills within the recreational use
range, with and without simultaneous alcohol intake have been reported [22, 23,24, 25].
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3. Illicit manuacture o piperazines
3.1 Illicit manufacture
The synthesis of BZP involves the reaction of piperazine and benzyl chloride, how-
ever, if piperazine as its free base is used, the reaction produces 1,4-dibenzylpipera-
zine (DBZP) as a by-product. The procedure shown in fgure I utilizing a mixture
of piperazine. HCl and piperazine hexahydrate proceeds with no formation of the
dibenzylated compound [26]. The reaction at 65oC produces the monohydrochloridesalt, which upon cooling and treatment with HCl forms the dihydrochloride salt.
The free base can be isolated by increasing the pH (> 12) and extracting with
chloroform. The synthesis is simple and rapid with a very high yield (84-85%). The
yield of the reaction can be increased to 95-96% and the reaction side products,
including 1,4-dibenzylpiperazine (DBZP) reduced by using a microwave method, inwhich the transformation of the microwave energy into heat leads to increased reac-
tion rates and higher yields [27].
Figure I. Synthesis o 1-benzylpiperazine (BZP).
NHHN
PhCH2Cl, EtOH
65oC
NH.HCl
N
NH.HCl
N EtOH, HCl
0oCNH.HCl
N
NH.HCl
N
NH
Naq. NaOH
HCl
HCl
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Recommended methods or the identifcation and analysis o piperazines in seized materials 11
There are several synthetic routes or 1-(3-chlorophenyl)piperazine (mCPP), the mostcommon o which is the reaction o diethanolamine with m-chloroaniline. Othermethods involve the reaction om-chloroaniline with bis(2-chloroethyl)amine or the
reaction o piperazine with m-dichlorobenzene. As with BZP, conventional synthesisroutes are simple and produce high yields (84-86%) [2, 28, 29, 30]. However, it isunlikely that the BZP, TFMPP or mCPP ound in illicit products have been synthe-sized in a clandestine laboratory as these compounds and their precursors are readilyavailable commercially. Indeed, ew piperazine clandestine laboratories have everbeen encountered with the most recently recorded in the literature being in 2008 inColorado, United States [29].
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4. Qualitative and quantitativeanalysis o materials containingpiperazines
Generally, in attempting to establish the identity o a controlled drug in suspectmaterial, the analytical approach must entail the determination o at least two uncor-related parameters, one o which should provide inormation on the chemical struc-ture o the analyte (or example, IR, MS; or tandem methods such as GC-MS).
It is recognized that the selection o these parameters in any particular case wouldtake into account the drug involved and the laboratory resources available to theanalyst. It is also accepted that unique requirements in dierent jurisdictions maydictate the actual practices ollowed by a particular laboratory.
4.1 Sampling
The principal reason or a sampling procedure is to permit an accurate and mean-ingul chemical analysis. Because most qualitative and quantitative methods used inorensic drug analysis laboratories require very small aliquots o material, it is vitalthat these small aliquots be representative o the bulk rom which they have beendrawn. Sampling should conorm to the principles o analytical chemistry, as laid
down, or example, in national pharmacopoeias or by regional or internationalorganizations. For general aspects o qualitative sampling o multi-unit samples,reer to the Guidelines on Representative Drug Sampling manual.
http://www.unodc.org/unodc/en/scientists/publications_manuals.html.
For seized material with obviously dierent external characteristics, a samplingmethod based on the Bayes model may be preerred over the hypergeometricapproach.
4.2 Solubility
The solubility properties provided in table 3 below can be utilized to separate
piperazines from diluents and adulterants [31]. For example, ether or acetone may
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Recommended methods or the identifcation and analysis o piperazines in seized materials 13
be used to separate BZP from 3-MeOPP and 2-MeOPP since neither of those two
compounds are very soluble in ether or acetone. BZP is also insoluble in water and
this property could be utilized to separate BZP from hydrochloride salts.
Compound: Acetone Chloroorm Ether Hexane Methanol Water
BZP VS PS FS VSS S I
TFMPP.HCl SS S VSS I FS VS
2-MeOPP.HCl I FS VSS I FS VS
3-MeOPP.2HCl I VSS VSS I S VS
4-MeOPP.2HCl VSS FS I I FS FS
Table 3. Solubility o selected piperazines [32]
Descriptive term Parts o solvent required or 1 part o solute
Very soluble (VS) Less than 1
Freely soluble (FS) From 1 to 10
Soluble (S) From 10 to 30
Sparingly soluble (PS) From 30 to 100
Slightly soluble (SS) From 100 to 1000Very slightly soluble (VSS) From 1000 to 10,000
Insoluble (I) More than 10,000
4.3 Screening tests
A screening test is a preliminary test which is used to indicate or eliminate a classor group o drugs. It also has the unction o narrowing the scope and ocusing thedirection o the analysis. By evaluating the results, urther tests are indicated whichcan lead to the conrmation o the identity o the unknown substance.
4.3.1 Colour tests
Colour or chemical spot tests are used in orensic drug analysis as a quick methodto give a presumptive indication o the possible presence or absence o a specicdrug or class o drugs in a questioned sample. The colour obtained in any particular
test may vary depending on the conditions o the test, amount o substance present,and extraneous material in the test sample. Colour tests are conducted by placing asmall amount o a sample into a spot plate cavity. A small amount o the particularreagent is then added, and any resulting colour change is observed. Colour testreagents must be checked with known substances when prepared, and a blank should
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14 Recommended methods or the identifcation and analysis o piperazines in seized materials
be run next to the sample to preclude alse positive results.
Colour tests are oten non-specic in nature and serve to include (or exclude) the
presence o a broad range o compounds. However, other colour reactions can bemore specic and demonstrate the presence or absence o certain unctional groups.By applying a series o dierent colour tests to the unknown sample, the analystcan narrow down the possible identity o the compound(s) present. It is mandatoryor analysts to conrm such results by the use o alternative techniques. Inormationon the preparation o the various reagents is shown below and the subsequent tablepresents the observed colour changes with various amounts o the dierent pipera-zines tested.
(a) Marquis reagent [33]:
Reagent A: 40 % Formaldehyde solutionReagent B: Sulphuric acid (conc.)
Method
Mix 1 drop o ormaldehyde solution with 1ml o concentrated sulphuric acid. Placethe test sample in a spot plate depression and add 3 drops o the mixed reagents.
(b) Simons reagent [33]:
Reagent A: 20% aqueous sodium carbonate solutionReagent B: 50% ethanolic acetaldehyde solutionReagent C: 1% aqueous sodium nitroprusside
Method
Prepared reagents should be stored in separate containers and rerigerated. Placethe test sample in a spot plate depression and add 1 drop o reagent A, ollowedby equivalent amounts o reagent B, then reagent C.
(c) Dragendor reagent [33]:
Reagent A: Bismuth subnitrate (1g)Reagent B: Hydrochloric acid (conc.)Reagent C: Ammonia (25%, aq)Reagent D: Potassium iodide (3g)Reagent E: Acetic acid (70%, aq)
Method
Dissolve 1g o bismuth subnitrate in a small amount o concentrated HCl. Add25% aqueous ammonia drop-wise until no more precipitate orms. Filter andpreserve the precipitate, wash it with water and then dissolve the precipitate in1 ml o concentrated HCl. Prepare a solution o 3 g potassium iodide in 1 mlwater. Add this to the precipitate solution. To the resulting solution, add 48 ml
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Recommended methods or the identifcation and analysis o piperazines in seized materials 15
o 70% aqueous acetic acid. Place the test sample in a spot plate depressionand add 3 drops o the reagent.
Interpretation of colour tests
When interpreting the results o a colour test, the analyst must keep two things inmind:
1. Is a colour observed ?2. O what signicance is the colour (or lack o colour) ?
Table 4. Piperazine colour test results. [33, 34]
Com-
pound
Marquis Conc.
H2SO
4
Simons Dragendorff
Sample 3 mg 10 mg 10 mg 3 mg 10 mg 3 mg 10 mg
BZP White to
brown-
green
precipi-
tate
with
fumes
White to
brown-
green
precipi-
tate
with
fumes
White to
dark
green
precipi-
tate
with
fumes
Pale
blue
Strong
Blue
Red
precipi-
tate
Red
precipi-
tate
2-MePP No
reaction
Not
tested
Not
tested
Blue Blue Red
precipi-
tate
Red
precipi-
tate
3-MePP No
reaction
Not
tested
Not
tested
No
reaction
No
reaction
Not
tested
Not
tested
4-MePP No
reaction
Not
tested
Not
tested
No
reaction
No
reaction
Not
tested
Not
tested
2-MeOPP No
reaction
Gradual
pink
colour
Gradual
pink
colour
Pale
blue
Blue Red
precipi-
tate
Red
precipi-
tate
4-MeOPP No
reaction
Fizz
no
colour
change
Fizz
no
colour
change
Pale
blue
Blue Red
precipi-
tate
Red
precipi-
tate
3-CPP/
mCPP
No
reaction
Fizz
no
colour
change
Fizz
no
colour
change
No
reaction
No
reaction
Red
precipi-
tate
Red
precipi-
tate
3-CPP.
HCl /
mCPP.HCl
Fizz
no
colourchange
Fizz
no
colourchange
Fizz
no
colourchange
Slight
purple
to blue
Blue,
slow to
yellow
Red
precipi-
tate
Red
precipi-
tate
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16 Recommended methods or the identifcation and analysis o piperazines in seized materials
4-CPP
HCl
Fizz
no
colourchange
Fizz
no
colourchange
Fizz
no
colourchange
Slight
purple
to blue
Blue,
slow to
yellow
Red
precipi-
tate
Red
precipi-
tate
3-TFMPP White to
pale
brown
precipi-
tate
White to
pale
brown
precipi-
tate
White
precipi-
tate,
with
fumes
No
reaction
Blue Red
precipi-
tate
Red
precipi-
tate
2-TFMPP White to
pale
brown
precipi-tate
White to
pale
brown
precipi-tate
White
precipi-
tate
No
reaction
Blue Red
precipi-
tate
Red
precipi-
tate
4-TFMPP Gradual
brown-
ish-red
colour
Gradual
brown-
ish-red
colour
Fizz
no
colour
change
No
reaction
Blue Red
precipi-
tate
Red
precipi-
tate
2-FPP Fizz
no
colour
change
Fizz
no
colour
change
Fizz
no
colour
change
Purple
to blue
Blue,
slow to
yellow
Red
precipi-
tate
Red
precipi-
tate
4-FPP Fizzno
colour
change
Fizzno
colour
change
Fizzno
colour
change
Blue Blue,slow to
yellow
Redprecipi-
tate
Redprecipi-
tate
Methafe-
tamine
HCl
Orange-
brown
Not
tested
Not
tested
Blue Not
tested
Red
precipi-
tate
Not
tested
MDMA
HCl
Black Not
tested
Not
tested
Blue Not
tested
Red
precipi-
tate
Not
tested
Dimethyl-
amfe-
tamine.
HCl
Brown Not
tested
Not
tested
No
reaction
Not
tested
Red
precipi-
tate
Not
tested
Table 4. (cont.)
Analytical Notes
Marquis reagent
This produces colour changes with a large number o heterocyclic compounds.However, as the sulphuric acid component o this reagent produces colourchanges when used alone, it is thereore essential to use sulphuric acid (3 drops)in the testing as a control.
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Recommended methods or the identifcation and analysis o piperazines in seized materials 17
4.4 Microcrystalline tests
Microcrystalline tests are chemical-precipitation tests that are quick, simple,extremely sensitive, and require only a small amount o sample. They are used toindicate the identity o a compound, or to determine its optical isomer.
These tests involve the ormation o crystals rom the reaction o the target com-pound with a reagent. The resulting crystals are analysed by means o a polarizingmicroscope and comparison with reerence material. Occasionally, it can be dicultto obtain an exact match between the sample and reerence material i, or example,other materials that may cause the deormation o crystals are present.
Microcrystalline tests can be perormed in the ollowing ways:
1. Direct addition: A portion o sample powder is placed on a microscopeslide and a drop o reagent is placed near it on the slide. The two are thendrawn together using a glass rod.
Example: Test or caeine using 5% gold chloride in dilute phosphoricacid.
For BZP-like compounds, the Marquis reagent showed negative results or aintcolouration. For most o the compounds the results are very similar to that o
the sulphuric acid control. For the purpose o comparison, the reagent producesa strong red-orange colour with amphetamines, while MDMA-type compoundsproduce a blue-black colour.
Simons reagent
A blue colour indicates the presence o a secondary amine and or some pipera-zines the colour changes gradually rom blue to yellow. Simons reagent is lesssensitive to BZP-like compounds than drugs such as methametamine or MDMA,thereore the result will be masked i these substances are also present.
The use o Simons reagent alone will do little to distinguish methamphetamineor MDMA rom piperazines, however, the combination o the Marquis reagentwith the Simons reagent may be eective in distinguishing some piperazines rommethametamine or MDMA while in the eld.
Dragendorff reagent
An orange, red-orange, or brown-orange precipitate suggests the presence o analkaloidal base and tertiary amines oten show a strong positive result. The resultswith the piperazines, while positive is not as strong as the result withdimethylametamine.
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18 Recommended methods or the identifcation and analysis o piperazines in seized materials
2. Solution mixing: A portion o sample powder is rst dissolved in a solvent(oten directly on the microscope slide itsel). A drop o reagent is placedbeside it and slowly drawn into the solution using a glass rod.
Example: Dissolution o a small quantity o a cocaine sample directly in20% acetic acid ollowed by the addition o 5% gold chloride.
3. Volatility or hanging drop tests: This technique is dependent upon the vola-tility o the compound being tested and is most requently applied to thedetermination o optical isomers o amines, particularly ametamine andmethametamine.
Example: A small amount o sample is placed in a spot plate and a dropo base added onto the sample. A drop o the test reagent is then placed
onto a cover slip and is inverted over the depression. Ater standing or5-10 minutes the resultant crystals can be observed.
4.4.1 Piperazine microcrystalline test (platinic bromide insulphuric acid)
Reagent
Dissolve 1 g platinic chloride (H2PtCl
66H
2O) in 1.7 ml HBr (40%). Dilute to 20
ml with 2 parts concentrated sulphuric acid and 3 parts water.
Method
Add reagent to an aqueous drop o test solution and evaporate.
Table 5. Results o piperazine microcrystalline tests using platinic bromide insulphuric acid [31]
Compound Crystal Produced
BZP Rectangles with indented ends
TFMPP Oils, then clusters of rods from centre core (bundles of rods),
(overgrown bow ties)
2-MeOPP Clusters of rods (wide blades/rods from core)
3-MeOPP Crosses with comb edges
4-MeOPP Rhomboid type crystals (rods/plates with rough edges)
4.4.2 Piperazine microcrystalline tests (mercury chloride)
Reagent
Aqueous solution o mercury chloride (10 g/L),
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Recommended methods or the identifcation and analysis o piperazines in seized materials 19
Method
An aliquot (10 l) of the test solution (1 g/L) is mixed with 10 l of the reagent
on a glass slide. A plastic pipette is used to aid nucleation and crystal formation [35].
Results
For BZP, transparent fat square plates were produced as shown in gure II, whileor TFMPP, a white precipitate was produced with no ormation o crystals.
Figure II. BZP mercuric chloride microcrystalline test [36]
4.5 Thin-layer chromatography (TLC)TLC is a commonly used technique or the separation and identication o illicitdrugs. It is inexpensive, rapid, sensitive (sub-milligram quantities o analyterequired), fexible in the selection o both the stationary and mobile phase, andamenable to a wide variety o substances, in base and salt orm, ranging rom themost polar to non-polar materials.
TLC plates (stationary phases)
Coating: Silica gel with layer thickness o 0.25 mm and containing an inert indica-tor, which fuoresces under UV light o wavelength 254 nm (Silica gel GF254).
Typical plate sizes: 20x20 cm; 20x10 cm; 10x5 cm (the latter should be used withthe 10 cm side vertical with the TLC tank).
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20 Recommended methods or the identifcation and analysis o piperazines in seized materials
Plates prepared by the analyst must be activated before use by placing them into
an oven at 120C for at least 10 to 30 minutes. Plates are then stored in a grease-
free desiccator over silica gel. Heat activation is not required for commercially
available coated plates.
Solvent systems
Prepare developing solvent system (system A, B, C, D or E as shown in table 6)as accurately as possible by use o pipettes, dispensers and measuring cylinders[33]. Leave the solvent system in the TLC tank or sucient time to allow vapourphase saturation to be achieved prior to the analysis (with adsorbent paper-linedtanks, this takes approximately 5 minutes).
Table 6. Solvent systems and visualization methods or TLC analysis opiperazines [33]
System Solvents Solvent proportions
(by ratio)
Visualisation method
A 2-butanone
dimethylformamide
aqueous ammonia (25%)
13
0.9
0.1
UV light
B 2-propanol
aqueous ammonia (25%)
95
5
Dragendorff reagent
C acetone
toluene
aqueous ammonia (25%)
20
10
1
Simons reagent
D methanol
aqueous ammonia (25%)
100
1.5
Iodoplatinate reagent
E 1-butanol
acetic acid
water
2
1
1
1% Iodine-methanol
Visualization methods
A. UV light
B. Dragendor reagent
Prepare as described in section 4.3.1.c.
C. Simons reagent (modication o reagent used in section 4.3.1.b).
Prepare solutions A (20% aqueous sodium carbonate solution) and B (1%aqueous sodium nitroprusside). Mix equal volumes o A and B and spraythe plate. Ater spraying the plates, expose them to acetaldehyde gas.
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Recommended methods or the identifcation and analysis o piperazines in seized materials 21
D. Iodoplatinate reagent
Solution A: aqueous 10% hydrogen hexachloroplatinate hexahydratesolution.
Solution B: aqueous 4% potassium iodide solution.
Mix solutions A, B and water in the ratio o 1 : 25 : 24 by volume.
E. 1% w/v Iodine-methanol solution
Spotting and developing
Apply as separate spots 1 L and 5 L aliquots o sample solution, 2 L o thestandard solutions and 2 L o solvent (as a negative control) on the TLC plate.Spotting must be done careully to avoid damaging the surace o the plate.
Visualization/detection
The plates must be dried prior to visualization. The solvent can be allowed toevaporate at room temperature or with a hot air blower. In the later case, care mustbe exercised that no component o interest is thermally labile. It is important orproper colour development that all traces o ammonia or other bases are removedrom the plate.
Analytical notes
The starting point o the run, i.e. the spotting line should be at least2 cm rom the bottom o the plate.
The spacing between applications o sample (spotting points) should be atleast 1 cm and spots should not be placed closer than 1.5 cm to the sideedge o the plate.
To avoid diuse spots during development, the size o the sample spot shouldbe as small as possible (2 mm) by applying solutions in aliquots rather thana single discharge.
Allow spots to dry and place plate into solvent-saturated tank (saturation othe vapour phase is achieved by using solvent-saturated pads or lter paper
as lining o the tank).
Remove plate rom the development tank as soon as possible ater thesolvent reaches the development line (10 cm rom starting line) markedbeorehand; otherwise, diused spots will occur.
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22 Recommended methods or the identifcation and analysis o piperazines in seized materials
Interpretation
Ater visualization, mark spots (e.g. by pencil) and calculate retardation actor (R)
values.
R=
Migration distance: rom origin to centre o spot
Development distance: rom origin to solvent ront
Table 7. Piperazine TLC Data [33]
Compound
Developing System (Rf)
A B C D E
BZP 0.03 0.15 0.13 0.25 0.66
2-TFMPP 0.11 0.41 0.36 0.33 0.8
mTFMPP 0.11 0.37 0.36 0.38 0.78
4-TFMPP 0.11 0.37 0.36 0.33 0.77
2-MeOPP 0.05 0.26 0.18 0.28 0.74
4-MeOPP 0.05 0.25 0.2 0.28 0.72
mCPP/3-CPP 0.11 0.38 0.37 0.32 0.77
4-CPP 0.07 0.3 0.3 0.27 0.77
2-FPP 0.12 0.4 0.36 0.28 0.74
4-FPP 0.07 0.3 0.25 0.24 0.74
Methamphetamine 0.09 0.37 0.32 0.21 0.76
Dimethylamphetamine 0.25 0.42 0.51 0.28 0.7
MDMA 0.09 0.36 0.32 0.21 0.74
Analytical notes
R
values are not always reproducible due to small changes in platecomposition and activation in solvent systems, tank saturation or develop-ment distance. Thereore, the R
values provided are indications o the
chromatographic behaviour o the substances listed.
It is essential that reerence standards be run simultaneously on the sameplate.
For identication purposes, both the R
value and the colour o the spotsater spraying with the appropriate visualization reagents should always beconsidered.
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Recommended methods or the identifcation and analysis o piperazines in seized materials 23
4.6 Gas chromatography (GC) with ameionization detection (GC-FID)
The GC instrument o choice or routine analytical work is the narrow bore capillarygas chromatograph, using columns with internal diameter between 0.2 and 0.32 mm.
4.6.1 Qualitative GC-FID method
GC oven conditions: Column temperature initially set at 100C and heldisothermal or 1 min., the temperature was then
ramped to 280C at 25C/min and held isothermalor 3 mins.
Column: 5% phenyl / 95% methyl silicone column, 10 mlength, 0.32 mm ID, 0.52 m lm thickness
Injection parameters: Mode: Split (50:1), 280C, 1 L injected
Carrier gas: Hydrogen 1.8 ml/min
Detector: FID, Detector temp: 280C
Table 8. Relative retention times (RRT) or samples dissolved in methanol
Compound Relative retention time (RRT)
Dimethyl sulfone 0.277
Methamfetamine 0.615
Dimethylphthalate 0.947
BZP 1.00 (4.212 min)
TFMPP 1.039
MDMA 1.043
2-MeOPP 1.155
4-MeOPP 1.287
3-MeOPP 1.303
Caffeine 1.362
4.6.2 Quantitative GC method
Internal standard stock solution
Prepare a solution containing 0.25 mg/ml dimethylphthalate in methanol.
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24 Recommended methods or the identifcation and analysis o piperazines in seized materials
Standard solution preparation
Prepare a solution by dissolving approximately 1.0 mg/ml o the piperazine to beanalysed in the internal standard stock solution.
Sample preparation
Accurately weigh an amount o the sample to be tested into a volumetric fask andll to the mark with the internal standard stock solution. I necessary, dilute thesample so the nal concentration is approximately that o the standard solutionconcentration.
GC oven conditions: Column temperature initially set at 130C and heldisothermal or 1 min, the temperature was thenincreased to 200C at 25C/min and held isothermalor 3 mins.
Column: 10 m x 0.32 mm x 0.52 m lm thickness
Phase: 5% phenyl/95% methyl silicone
Carrier gas: Hydrogen at 1 ml/minute
Injection Parameters: Split (50:1), 280C, 1 L injection
Detector: FID
Results
Linear range: 0.050-1.206 mg/ml
Repeatability: RSD less than 0.5%
Correlation coecient: 0.999
Accuracy: Error less than 5%
Table 9. RRT or samples dissolved in internal standard stock solution
Compound Relative retention time (RRT)
Methamphetamine 0.472
2-MeOPP 1.279
BZP 1.00 (2.23)
TFMPP 1.073
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Recommended methods or the identifcation and analysis o piperazines in seized materials 25
3-MeOPP 1.506
Dimethylphthalate 0.917
Caffeine 1.969
4-MeOPP 1.547
4.7 Gas chromatography-mass spectrometry(GC-MS)
GC-MS is one o the most commonly used techniques or the identication oorensic drug samples. As a hyphenated technique, it unies the separation powerand sensitivity o a GC with the analyte specicity o a spectroscopic technique. Itcan provide highly specic spectral data on individual compounds in a complexmixture without prior isolation.
4.7.1 GC-MS method 1 [37]
GC oven conditions: Column temperature initially set at 100C and heldisothermal or 5 mins., the temperature was thenramped to 290C at 10C/min and held isothermal or20 mins.
Column: 5% phenyl/95% methyl silicone column, 30 m length,0.25 mm ID, 0.25 m lm thickness
Injection parameters: Splitless, 1 L injected
Injector temp: 250C
Carrier gas: Helium, 1.1 ml/min
Detector: Ionization mode: EI mode, 70 eV
Transer line temp: 290C
Ion source temperature: 200C
MS parameters Scan parameters: TIC
Scan range: 30-350 amu
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26 Recommended methods or the identifcation and analysis o piperazines in seized materials
Table 10. GC RT and RRT or samples dissolved in methanol
Compound GC RT GC RRT
4-FPP 13.37 0.97
2-MeOPP 14.85 1.08
1-Phenylpiperazine 13.75 1.00
3-MeOPP 16.47 1.20
3-FPP 13.71 1.00
4-MeOPP 16.15 1.17
2-FPP 12.77 0.93
2-CPP 14.64 1.06
3-CPP/mCPP 15.99 1.16
4-CPP 16.04 1.17
2,3-XP 15.26 1.11
3,4-XP 16.25 1.18
mTFMPP 14.65 1.07
2,5-XP 14.81 1.08
2,4-XP 14.87 1.08
2-TFMPP 13.53 0.98
3-TMP 13.32 0.97
MBZP 13.05 0.95
BZP 13.10 0.95
MDBZP 17.32 1.26
2-PEP 15.00 1.09
Note: Relative retention times were calculated rom data in table 3 in reerence 37
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Recommended methods or the identifcation and analysis o piperazines in seized materials 27
4.7.2 GC-MS method 2 [38]
GC oven conditions: Column temperature initially set at 80C and held
isothermal or 4 mins., the temperature was thenramped to 280C at 20C/min, held isothermal or 8mins. The temperature was then increased to 290C at20C/min and held isothermal or 11.5 minutes.
Column: Shimadzu QP2010 GC/MS with a HP5MS column (30 mx 0.25 mm, 0.50 m)
Injection parameters: Mode: Splitless, injection temperature (225C) 1 Linjected
Carrier gas: Helium, 1 ml/minute. Pressure (9.5 psi)
Detector: Ionization mode: EI mode, 70 eV
Transer line temp: 300C
Ion source temperature: 230C
MS parameters: Solvent delay: 3 mins.
Scan parameters: TIC
Scan range: 40-450 amu at 1 scan/sec
Table 11. RT and RRT or samples dissolved in methanol
Compound RT (mins) RRT (mins)
Quinoline 9.049 0.82
BZP 10.989 1.00
4-FPP 11.132 1.01
TFMPP 11.185 1.02
3-MePP 11.800 1.07
4-MePP 11.800 1.07
2-CPP 11.867 1.08
2-MeOPP 11.861 1.08
mCPP 12.600 1.15
4-MeOPP 12.639 1.15
4-CPP 12.639 1.15
Pyribenzamine 13.941 1.27
DBZP 14.919 1.36
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28 Recommended methods or the identifcation and analysis o piperazines in seized materials
4.7.3 GC-MS method 3 [39, 40]
Sample preparation: Samples dissolved in acetonitrile.
GC oven conditions: Column temperature initially set at 100C and heldisothermal or 1 min., the temperature was thenramped to 180C at 12C/min. and held isothermal or2 mins. The temperature was then increased to 200Cat 10C/min and held isothermal or 5 mins.
Column: Agilent 7890A GC/MS with a 100% trifuoropropylmethyl polysiloxane (Rtx-200) capillary column (30 m x0.25 mm, 0.50 m)
Injection parameters: Mode: Splitless, 250C, 1 L injected
Carrier gas: Helium, 0.7 ml/minute. Pressure (10 psi)
Detector: Ionization mode: EI mode, 70 eV
Transer line temp: 280C
Ion source temp: 230C
Interace temp: 250C
4.8 Gas chromatography-inrared detection(GC-IRD) [39, 40]
Recent advances in Fourier transorm inrared (FTIR) spectroscopy and capillarygas chromatography have made it possible to produce hyphenated GC-FTIRinstruments. This technique uses the properties o capillary gas chromatography tovaporize and separate the individual components o a sample ollowed by thedetection o vapour phase inra red spectra o each component.
Sample preparation: Samples dissolved in acetonitrile.
GC-IRD operating conditions
GC oven conditions: Column temperature initially set at 100C and heldisothermal or 1 min, the temperature was thenramped to 230C at 20C/min and held isothermal or15 mins.
Column: HewlettPackard 5890 Series II GC with a 50%
phenyl50% methyl polysiloxane (Rxi-50) capillarycolumn (30 m x 0.25 mm (i.d.), 0.5 m
Injection parameters: Mode: Splitless, 1L
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Recommended methods or the identifcation and analysis o piperazines in seized materials 29
4.9 High pressure liquid chromatography (HPLC)
In addition to GC, HPLC is another major separation technique used in orensicdrug analysis. Reverse phase chromatography is most commonly used or the analy-sis o drugs in seized materials and the most universal and versatile column is abonded octadecyl silica column (C18). Column length, diameter, particle size, poresize and carbon load should be considered beore nal selection o the column. Asthere are a large variety o stationary and mobile phases available to the analyst,all methods must be properly validated and/or veried prior to routine use.
4.9.1 HPLC method 1 (qualitative) [37]
Sample preparation
Dissolve approximately 10 mg o sample in 10 ml o 20 mM HCl:methanol, 1:1solution. Dilute 1 ml o the solution with methanol to a total volume o 10 ml.Filter with a 0.45 m membrane lter.
Column: 4.6 mm (ID) x 250 mm, 5 m, C18 thermostated at 40C.
Mobile phase: (A) Acetonitrile(B) 5mM Heptafuorobutyric acid
A gradient program was utilized
0 mins 18:82, A:B
10 mins 18:82, A:B
25 mins 28:72, A:B
50 mins 30:70, A:B
Flow rate: 1 ml/min
Injection volume: 10 L
Detection: Photo diode array (PDA) 199-360nm
Carrier gas: Helium at 0.7 mL/min., pressure (10 psi )
IR Range 4000-650 cm-1
Resolution 8 cm-1
Detection: scan rate Scan rate: 1.5 scans/sec
IRD fow cell: 280C
Transer line temp: 280C
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Table 12. RT and RRT to 1-phenylpiperazine or selected piperazines
Compound Relative time (RT) RRT
4-FPP 4.20 0.68
2-MeOPP 8.02 1.29
1-Phenylpiperazine 6.21 1.00
3-MeOPP 9.50 1.53
3-FPP 10.82 1.74
4-MeOPP 8.02 1.29
2-FPP 9.42 1.52
2-CPP 16.15 2.60
3-CPP/mCPP 19.03 3.06
4-CPP 19.38 3.12
2,3-XP 26.63 4.29
3,4-XP 22.26 3.58
4-TFMPP 31.66 5.10
2,5-XP 29.41 4.74
2,4-XP 30.83 4.96
TFMPP 30.06 4.84
3-TMP 5.73 0.92
MBZP 6.80 1.09
BZP 6.60 1.06
MDBZP 7.05 1.14
2-PEP 8.12 1.31
Note: RT and RRT values calculated rom data in Table 3 in reerence 37
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Recommended methods or the identifcation and analysis o piperazines in seized materials 31
4.9.2 HPLC method 3 (qualitative and quantitative) [31]
Sample preparation
Accurately weigh an amount o sample into a volumetric fask and dilute with0.01M HCl. I necessary, dilute the sample so the nal concentration approximatesthe standard concentration.
HPLC operating conditions
Column: 4.6 mm x 250 mm, 10 m, C18
Mobile phase: 86% Sodium hexylammonium phosphate (NaHAP) Buer :14 % acetonitrile.
Buer preparation (4000 ml distilled water, 10 g sodiumhydroxide, 30 ml phosphoric acid and 8 ml hexylamine)
Flow rate: 1 ml/min
Injection volume: 3 L
Detection: UV, 210 nm
Results
Linear range: 0.051-0.508 mg/ml
Repeatability: RSD less than 3%
Correlation coecient: 0.9993
Accuracy: error less than 5%
Table 13. Relative retention times o selected piperazines
Compound RRT
2-MeOPP 1.0 (5.13min)
BZP 0.45
3-MeOPP 1.10
4-MeOPP 0.87
TFMPP 5.11
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4.10 Capillary electrophoresis (CE)
Capillary electrophoresis is an analytical technique involving the separation o
charged species based on their migration under the infuence o an applied electriceld through a used silica capillary. The ollowing section presents a method orboth the qualitative and quantitative analysis o selected piperazines using capillaryelectrophoresis (CE).
4.10.1 CE method (qualitative and quantitative) [31]
Internal standard stock solution
Prepare a solution o thiamine hydrochloride in water at a concentration o 0.2 mg/ml.
Standard solution preparation
Prepare a standard solution at approximately 0.4 mg/ml dissolving in the internalstandard stock solution.
Sample preparation
Accurately weigh an amount o sample and dissolve with internal standard stocksolution. The sample should then be diluted with internal stock solution to a con-centration approximately equal to that o the standard.
Mode: Free zone
Capillary: 34 cm x 50 m used silica capillary
Run buer: 100 mM lithium phosphate buer at pH 2.3
Detector: UV, 210 nm
Voltage: 20 kV
Temperature: 20C air cooled
Injection: Hydrodynamic, 50 mbar or 2.5 s
Run time: 6 mins.
Rinse time: 1 min.
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Recommended methods or the identifcation and analysis o piperazines in seized materials 33
Results
Linear range: 0.05-1.2 mg/ml
Repeatability: RSD less than 3%
Correlation coecient: 0.999
Accuracy: error less than 5%
Table 14. Relative migration times (RMT)
Compound RMT Thiamine 0.892
BZP 1.0 (3.525 mins)
TFMPP 1.417
2-MeOPP 1.337
3-MeOPP 1.349
4-MeOPP 1.296
4.11 Fourier transorm inrared (FTIR)spectroscopy
The conrmation o the identity o a substance can be achieved by FTIR. Unequivo-cal identication o a particular piperazine is thus possible rom each unique spec-trum. For powders, considered rom prior chromatographic analysis to be reasonablypure, the inrared spectrum o the powder may be run directly in a KBr disc orcomparison with those o the ree base or HCl salt o a particular piperazine. For
tablets, capsules and mixtures o powders, an extraction procedure would be requiredto liberate the ree base in pure orm.
Analytical notes
The KBr disc method consists o grinding a dry sample to a very ne powder,then mixing about 2 mg o homogenized sample powder with 200 mg ocareully dried and ground KBr. Ater grinding, the mixture is pressed into athin transparent disk.
KBr should be IR Grade and dried at 105C or a minimum o one hour.It can be stored in a desiccator containing a strong desiccant (silica gel) orlet in the oven and removed when required.
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Table 15. Characteristic IR spectral bands or selected piperazines (liquidsamples were analysed as thin flms between NaCl plates and solids wereanalysed as KBr discs, scan range 600 cm-1-4000 cm-1) [33].
Substance Characteristic IR bands (wavenumber, cm-1)
BZP 698, 739, 1142, 1319, 1454
TFMPP 1120, 1163, 1319, 1354, 1450
2-TFMPP 1036, 1109, 1136, 1315, 1454
4-TFMPP 1068, 1109, 1244, 1325, 1614
2-MeOPP 748, 1028, 1240, 1450, 1500
BZP.2HCl 702, 748, 957, 1074, 1431
TFMPP.HCl 1120, 1165, 1321, 1352, 1589
4-MeOPP.2HCl 835, 1018, 1255, 1444, 1518
4-CPP.HCl 818, 1147, 1253, 1454, 1497
4-FPP.2HCl 845, 1165, 1228, 1423, 1512
2-FPP.HCl 764, 1149, 1209, 1252, 1500
3-CPP/mCPP.HCl 750, 945, 1253, 1489, 1595
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5. Library inormation
There are ew libraries available or the identication o the piperazine compoundsand i available are costly. Takahashi et al. outlines a method or the creation osuch a library [37].
5.1 Ultraviolet (UV) spectrophotometry
Selected piperazines in aqueous acid have absorbance maxima at the ollowingwavelengths.
Table 16. UV Spectroscopy data or selected piperazines [31]
Compound Maximum Absorbance (nm)
BZP 193
2-MeOPP 206
3-MeOPP 210
4-MeOPP 196
TFMPP 202
5.2 GC-MS data or selected piperazinesThe ragmentation patterns o selected piperazines were obtained using electronionization (EI) at an energy o 70 eV. The ions are listed in decreasing order opeak intensity under the experimental conditions used.
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Table 17. Characteristic ions o the EI mass spectra or selectedpiperazines [33]
Substance Characteristic ions (m/z)
BZP 91, 134, 56, 120, 176 (M+)
mTFMPP 188, 145, 172, 56, 230 (M+)
oTFMPP 188, 145, 172, 56, 230 (M+)
pTFMPP 188, 145, 172, 56, 230 (M+)
oMeOPP 150, 135, 120, 192 (M+)
pMeOPP 150, 135, 120, 192 (M+)
mCPP 154, 56, 196 (M+)
pCPP 154, 56, 196 (M+)
oFPP 138, 122, 56, 180 (M+)
pFPP 138, 122, 56, 180 (M+
)
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Aitchison L.,Exposure to Benzylpiperazine (BZP) in Adolescent Rats: Adulthood inAnxiety-like Behaviour. Masters. University o Canterbury, 2006.
Antia U., Lee H.S., Kydd R.R., Tingle M.D. and Russell B.R., Pharmacokinetics oParty Pill Drug N-Benzylpiperazine (BZP) in Healthy Human Participants, Foren-sic Science International, 186 (1-3) (2009) 6367.
Baltzly R., Buck J.S., Lorz E. and Schon W., The Preparation o N-Mono-Substitutedand Unsymmetrically Disubstituted Piperazines, Journal o the American Chemical
Society, 66 (2) (1944) 263266.
Bishop S. C., Advanced Capillary Electrophoretic Techniques or the Detection oDate-Rape and Club Drugs or a Forensic Setting, Ph.D. Ohio University, 2004.
Bossong M. G., Brunt T. M., Van Dijk J. P., Righter S.M., Hoek J., GoldschmidtH.M.J. and Niesink R.J.M., mCPP: an undesired addition to the ecstasy market,
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Bossong M.G., Van Dijk J. P and Niesink R.J.M., Methylone and mCPP, Two New
Drugs o Abuse? Addiction Biology, 10 (2005) 321-323.
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Bryson-Hammond K.A., Recreational Drug Using Behaviour and Legal Benzyl-piperazine Party Pills. Ph.D. Victoria University o Wellington, (2008).
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Herndon J.L., Pierson M.E. and Glennon R.A., Mechanistic Investigation o theStimulus Properties o 1-(3-Trifuoromethylphenyl)Piperazine, Pharmacology Bio-chemistry and Behaviour, 43 (1992) 739-748.
Johanson C.E., Kilbey M. Gatchalian K. and Tancer M., Discriminative StimulusEects o 3,4-methylenedioxymethametamine (MDMA) in Humans Trained to Dis-criminate Among d-Ametamine, meta-chlorophenylpiperazine and Placebo, Drugand Alcohol Dependence, 81 (2006) 2736.
Machado A., Tejera E. and Rebelo I., Infuence o Arylpiperazines Aromatic Struc-ture Over Dierential Anity or 5-Ht1a and 5-Ht2a Receptors, Journal o Bio-medicine, 2 (2009) 9-19.
Maurer H.H.F., Select Benzyl- and Phenyl- Piperazine Designer Drugs, MicrogramJournal, 2 (1-4) (2004) 22-26.
Moloney G.P., Garavelas A., Martin G.R., Maxwell M. and Glen R.C., Synthesisand Serotonergic Activity o Variously Substitute (3-Amido)Phenylpiperazine Deriv-
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Nikolova I. and Danchev N., Piperazine Based Substances o Abuse: A New PartyPills on Bulgarian Drug Market, Biotechnology & Biotechnological Equipment, 22(2) (2008) 652-655.
Patent # WO 2008/043839, Aryl Piperazine Derivatives Useul or the Treatment o
Neuropsychiatry Disorders.Peters F.T., Schaeer S., Staack R.F., Kraemer T. and Maurer H.M., Screening orand Validated Quantication o Ametamines and o Ametamine and Piperazine-Derived Designer Drugs in Human Blood Plasma by Gas Chromatography/MassSpectrometry, Journal o Mass Spectrometry, 38 (2003) 659676.
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